E. coli MutY and its mammalian homologues (MYH) play an important role in the prevention of mutations associated with 7,8-dihydro-8-oxo-2'-deoxyguanosine (OG) by removal of misincorporated adenine residues from OG:A mismatches. Recently, a direct correlation between mutations in the gene encoding human MutY (hMYH) and colorectal cancer has been uncovered. This highlights the importance of base-excision repair, and the repair of OG:A mismatches in the prevention of carcinogenesis. My laboratory has developed a rigorous and multifaceted research program aimed at providing a detailed understanding of the functional properties of MutY and hMYH. Importantly, our functional analysis of hMYH variants has been important for establishing the connection to colorectal cancer. In order to further the understanding of the relationship between hMYH and colorectal cancer, as well as continue our understanding of the complex features of mismatch recogntion by this unique BER glycosylase, we propose the following in this research grant application: (1) We will determine the functional properties of variants of hMYH that are correlated with colorectal cancer. Specifically, this will involve analyzing the repair of OG:A mismatches by a representative set of hMYH variants in bacterial and mammalian cells. (2) We will evaluate the adenine glycosylase activity of this same representative set of hMYH variants to determine if reduced OG:A repair is due to defects in the intrinsic adenine removal activity of the enzyme. Aims 1 and 2 will provide important information on the relationship of these variants to colorectal cancer. (3 ) We will provide insight into specific steps that are involved in OG:A mismatch recognition and adenine removal by MutY using fluorescence spectroscopy with substrates and substrate analogues. This will allow us to reveal the important features of OG and A that are involved in initial base-pair recognition, and how recognition of OG is coupled to adenine extrusion and removal. This aspect will utilize substrate analogues that we have characterized previously and build upon the kinetic framework we have developed for analyzing MutY. (4) We will provide insight into the mechanism of adenine excision by examining the structural properties of Bacillus stearotheromophilus MutY with transition state and substrate analogue-containing duplexes using X- ray crystallography.